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No description available.

The optimisation of recombinant protein production by animal cell cultures is important for the economic feasibility of these processes. Simultaneously with product yield, product authenticity is a crucial aspect to consider as it may per se affect the therapeutic value of such proteins. More defined culture media are being developed, particularly to ensure batch product consistency. A Chinese Hamster Ovary cell line (CHO 320) producing human interferon-γ (IFN-γ), a glycosylated protein, was chosen to investigate the effects of the culture environment on (I) cell growth, (2) product yield and (3) product authenticity. A statistical approach was used to identify important culture components for cell growth and IFN-γ production. When the concentration of the resulting positive variables was initially increased in culture, improvements of approximately 40% in both of these parameters were achieved; the glycosylation of IFN-γ was not affected. The former analysis also indicated that different stimuli were required for growth and production. Fed-batch feeding of glucose and glutamine, components depleted early from culture, did not prolong cell growth or IFN-γ production but the initial glycosylation pattern of IFN-γ was a function of glutamine concentration. Bovine serum albumin (BSA) was shown to have important role(s) in culture and cell growth was not possible in its absence. Pluronic F68, alone or in combination with a lipid mixture or linoleic acid, was able to restore cell growth in low BSA (1 mg/ml) cultures. However, IFN-γ production was significantly reduced and the extent of IFN-γ glycosylation also changed. These effects were related to: (1) BSA concentration, (2) BSA type, and ultimately, (3) lipid composition of the culture. The results reported in this thesis exhibit the necessity to consider the effects of the culture environment not only on cell growth and product yield but also on product authenticity throughout any optimisation process.

No description available.

No description available.

No description available.

The binding of a drug or other ligand to plasma proteins can effect their absorption, metabolism and excretion which can lead to a change in its toxicity and therapeutic action. Fluorescence is a technique that has been used to study such interactions and has the advantages of extreme sensitivity and specificity. Previously fluorescence has been monitored in the UV /vis range of the spectrum. However, a new development is long wavelength fluorescence (600-1000nm), which has the added benefits of a lower background, decreased scattering, decreased photodecomposition and the availability of inexpensive, solid state, optical components. Certain dyes including polymethines, xanthenes and phenoxazines that fluoresce in the long wavelength region (600-1000nm) of the spectrum were investigated for use as fluorescence probes. Nile Red, a strongly hydrophobic phenoxazine dye, was found to have an emission wavelength and intensity which was strongly dependent on the polarity of its environment. Consequently, it was bound to certain proteins including bovine and human serum albumin, aI-acid glycoprotein and B-lactoglobulin and provided both qualitative and quantitative information on the nature and type of binding site on the protein. It was also used in the study of ligand protein binding interactions in which competition for a binding site on the protein occurs between the probe and other ligands such as drugs or fatty acids. The project also involved a preliminary investigation into a novel double probe technique for the study of drug-protein interactions and the development of a flow injection analysis method involving gradient titration of a drug against a probe:protein system.

I dagens samhälle finns en stor marknad för proteintillskott. Kosttillskottsföretagen gör stora pengar för att atleter tror sig dra nytta av tillskotten och pulvren. Är proteintillskott i form av vassle, kasein, soja och kreatin att föredra eller är det vanlig kost som är bäst för att bygga muskler i samband med träning? Syftet med denna ämnesfördjupning är att fokusera på proteiner som näringsämne samt som ett tillskott får både erfarna och icke-erfarna styrkeidrottare samt en jämförelse mellan olika typer av proteintillskott. Ämnesfördjupningen försöker också att svara på vilket tillskott som är att föredra? När och hur mycket som skall intas för bästa effekt? Samt om en ökad proteinkonsumtion kan få konsekvenser för konsumenten?

p53 is a tumour suppressor protein that is stabilised and activated by DNA damage. DNA damage-induced p53 is able to bring about either cell cycle arrest or apoptosis by the induction of p53-responsive genes such as mdm2 and p21 waf-I. Mdm2 regulates p53 function by blocking the transcriptional transactivation domain of p53 and also by targeting p53 for degradation via an ubiquitin-mediated pathway. Increases in the levels and activity of p53 are brought about by post-translational modifications. The most widely studied modification of p53 is phosphorylation, mediated by several DNA damageactivated kinases. Poly(ADP-Ribose) Polymerase-l (PARP-l) is also a DNA damageactivated enzyme which covalently modifies several target proteins by poly(ADPribosylation). It is well established that PARP-1 plays a key role in DNA base excision repair. More recently, several studies have implicated PARP-1 in the regulation of p53 function in response to DNA damage, although the nature of this relationship has been controversial. This study aimed to clarify and investigate further the role of PARP-1 in p53 regulation using PARP-1 proficient and PARP-1 deficient mouse embryonic fibroblasts (MEFs) as well as a novel potent PARP-1 inhibitor (AGI4361; Ki < 6nM). In this study, both primary and immortalised PARP-l MEFs were used. Initial experiments revealed a tendency for PARP-l +/+ MEFs to develop p53 mutations during immortalisation. Interestingly. PARP-1 -/- MEFs retained wild-type p53, suggesting that the absence of PARP-l bypasses the requirement for p53 to be mutated during the immortalisation of MEFs. As these cells could not be used to analyse p53 responses, experiments were perfonned on primary PARP-l MEFs. However. the primary PARP-l- - MEFs were found to grow very slowly compared to their PARP-1 proficient counterparts. Interestingly. treatment of primary PARP-1+1+ MEFs with AG14361 had a similar effect on cellular growth. This growth inhibition in the absence of PARP-1 was only evident in primary and not immortalised cells. It was therefore decided to stably transfect immortalised PARP-l-- MEFs, expressing wild-type p53, with a plasmid construct containing PARP-l to produce an isogenic cell line pair. These cells have been used, together with a human colorectal carcinoma cell line (HCT-116) and the potent PARP-1 inhibitor AG14361 to analyse the p53 response to different DNA damaging agents. In response to ionising radiation and ultra violet radiation, the absence of PARP-1 did not alter the induction or activity of p53. In response to the alkylating agent temozolomide, treatment of PARP-l proficient MEFs with AG14361 potentiated the increase in p53 protein levels without affecting the transcriptional transactivation activity of p53, possibly due to an impaired repair of the DNA damage and hence increased signalling to p53 due to the persistence of DNA strand breaks. However, similar results were not obtained in the absence of PARP-1 protein (P ARP-1-/- MEFs) or in HCT -116 cells treated with AG 14361 The data presented do not support the hypothesis that PARP-1 is directly involved in the DNA damage induced regulation of p53. There may, however, be an altered p53 response in the absence of PARP-l when cells are treated with particular DNA damaging agents, due to an impaired DNA repair pathway.

Studies of the purification, characterization and primary structure of protein inhibitors of trypsin and -amylase from seeds of Job's Tears (Coix lachryma-jobi) were undertaken. The major trypsin inhibitor from seeds of Coix was purified by heat treatment, fractional precipitation with ammonium sulphate, ion-exchange chromatography, gel filtration and preparative reversed-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S- carboxymethylated protein by digestion with trypsin, chymotrypsin and the S.aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong similarity with a number of Bowman-Birk inhibitors from legume and cereal seeds. A protein inhibitor of locust gut ζ-amylase was purified from seeds of Coix using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two identical or closely similar subunits of M(_r) about 26 400. These two isomers also had very similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, B. subtilis. A. oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of similarity with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean). Endochitinase activity was demonstrated by following the release of radioactivity from [(^3)H] chitin. As far as can be ascertained from the literature this is the first characterization of a plant protein with activity as an enzyme and as an enzyme inhibitor. Preliminary molecular studies were also carried out, including the isolation and in vitro translation of mRNA fractions from developing seeds of Coix.